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Follicular Fluid Hormonal and Non-Hormonal Composition After Gnrha

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Follicular Fluid Hormonal and Non-Hormonal Composition After Gnrha Follicular fluid hormonal and non-hormonal composition after GnRHa Claus Yding Andersen, Professor, MSc, DMSc Laboratory of Reproductive Biology University Hospital Copenhagen, University of Copenhagen Copenhagen, Denmark E-mail: [email protected] ESHRE Campus “GnRH Agonist for Triggering of Final Oocyte Maturation Time for a Paradigm Shift” Madrid, Spain – 3 December 2010 Outline Effects of the mid‐cycle surge of gonado‐ tropins on the preovulatory follicle Follicular fluid hormone profile from GnRHa and hCG induced patients Profile of factors in follicular fluid from GnRHa and hCG induced patients of importance for oocyte maturation ESHRE Campus “GnRH Agonist for Triggering of Final Oocyte Maturation Time for a Paradigm Shift” Madrid, Spain – 3 December 2010 GnRH agonist used for ovulation induction in women following a GnRH antagonist protocol GnRH antagonists protocols allows ovulation to be induced with either hCG or GnRH agonists GnRHa induces an endogenous flare‐up of gonadotropins but also a pituitary down‐regulation resulting in an attenuated surge of gonadotropins The midcycle surge of gonadotropings: GnRHa versus the natural cycle hCG GnRHa 14h Natural 4h 20h 14h 0 20 h 48h Hoff et al., 1983 Itskovits et al., 1991 Differences between GnRH agonist and hCG induced trigger of ovulation Duration and amplitude of the GnRHa induced ovulation is a lot shorter The total LH‐like activity in hCG stimulated ovulation induction is a lot higher The GnRHa induced ovulation produces a combined FSH and LH surge Main physiological functions of the midcycle surge of gonadotropins Induce resumption of meiosis of the enclosed oocyte Induce the ovulatory process itself and expulsion of oocyte Prepare the function of the corpus luteum in order to sustain the luteal phase These functions have different sensitivity towards the amplitude and composition of the midcycle surge of gonadotropins – at least in rodent models! Sensitivity of preovulatory follicles to gonadotropins At around 30 % of the preovulatory surge meiotic resumption is induced At around 85 % of the preovulatory surge ovulation itself is induced FSH induces LHR formation in the developing corpus luteum Mattheij JA et al., Eur J Endocrinol, 1995;132:91 Vermeiden JPW and Zeilmaker GH, Endocrinol, 1974; 95:341 Does GnRH agonist for triggering of ovulation induce changes in the preovulatory follicle different from those of hCG? And to what extent does a possible change affect oocyte maturation and implantation potential? Characteristics of follicular fluid from women receiving either Buserelin or hCG to induce ovulation Claus Yding Andersen1, P Humaidan2, H Ejdrup Bredkjær3, L Bungum2, M Bungum2, ML Grøndahl3, J Ingemanssen3 & LG Westergaard4 1Laboratory of Reproductive Biology, Section 5712, University Hospital of Copenhagen, Copenhagen, Denmark, 2The Fertility Clinic, Skive Sygehus, Skive, Denmark 3The Fertility Clinic, Hvidovre Hospital, Hvidovre, Denmark 4The Fertility Clinic Trianglen, Lundevangsvej 12, Hellerup, Denmark Yding Andersen et al., Hum Reprod, 21, 2006 Aim • To compare hormonal profiles of follicular fluid from women undergoing IVF/ICSI following a flexible GnRH antagonist protocol – randomized to final oocyte maturation with either hCG or a single bolus of GnRHa (Buserelin) Open label prospective randomised two‐centre study FF from our original study in which the reproductive outcome was poor Serum concentrations of oestradiol and progesterone (nmol/l) in GnRHa versus hCG‐group (mean ±SD) Day Oestradiol Progesterone Buserelin hCG Buserelin hCG Ovulation 7.1 ± 4 6.4 ± 3 5.5 ± 5.8 5.0 ± 3.0 induction OPU 4.2 ± 2 4.4 ± 2 28 ± 18c 49 ± 33c OPU + 7 days 2.9 ± 2a 7.1 ± 4a 39 ± 30d 283 ± 205d OPU + 14 days 2.7 ± 2b 5.6 ± 5b ND ND abcd: P<0.001 Student t‐test Hormonal profile of follicular fluid from women receiving either Buserelin or hCG to induce ovulation Number of samples Agonist hCG 64 (32) 74 (37) Neg. hCG Pos. hCG Neg. hCG Pos. hCG 44 (22) 20 (10) 44 (22) 30 (15) Hormonal profile of follicular fluid from women receiving either Buserelin or hCG to induce ovulation Agonist hCG FSH LH hCG FSH LH hCG 6,3 ± 0,6 11,1 ± 0,6 ND 3,3 ± 0,2 3,6 ± 0,3 139 ± 8 M II oocytes : 84 % M II oocytes : 63 % No significant differences between pregnant and non‐pregnant women Hormonal profile of follicular fluid from women receiving either Buserelin or hCG to induce ovulation Agonist hCG Oestradiol (μmol/l) 1,9 ± 0,2 1,8 ± 0,2 Neg. hCG Pos. hCG Neg. hCG Pos. hCG 1,9 ± 0,2 1,9 ± 0,9 1,9 ± 0,2 1,7 ± 0,2 Progesterone (μmol/l) 70 ± 4* 90 ± 6* Neg. hCG Pos. hCG Neg. hCG Pos. hCG 73 ± 5** 48 ± 5** 86 ± 4 90 ± 6 Data: Mean ±SEM Hormonal profile of follicular fluid from women receiving either Buserelin or hCG to induce ovulation Agonist hCG Inhibin‐A (ng/ml) 37,4 ± 4,8 40,1 ± 3,1 Neg. hCG Pos. hCG Neg. hCG Pos. hCG 44,7 ± 7,1* 24,8 ± 2,3* 37,6 ± 4,3 43,6 ± 5,1 Inhibin‐B (ng/ml) 35,6 ± 2,8 40,1 ± 3,1 Neg. hCG Pos. hCG Neg. hCG Pos. hCG 35,3 ± 3,3 36,7 ± 5,5 39,2 ± 4,2 41,3 ± 4,7 Data: Mean ±SEM Conclusions The poor reproductive outcome of inducing ovulation by a GnRH agonist does not seem to be caused by inappropriate intrafollicular oocyte maturation Despite significant differences in FF levels of progesterone they were too small to explain a difference On the contrary more oocytes may actually reach M II What about factors affecting oocyte maturation directly and substances that affect the corpus luteum function. Yding Andersen et al., Hum Reprod, 21, 2006 Levels of the EGF‐like peptide amphiregulin are significantly reduced in follicular fluid after GnRHa triggering of final oocyte maturation P.Humaidan 1, L.G.Westergaard 2, AL.Mikkelsen 3, M. Fukuda 4 and C.Yding Andersen 5 1The Fertility Clinic, Skive Regional Hospital (Viborg), Denmark , 2 The Fertility Clinic, Odense University Hospital, Denmark 3 The Fertility Clinic, Holbæk Hospital, Denmark,, 4 M and K Health Institute, Ako, Japan 5 Laboratory of Reproductive Biology, University Hospital of Copenhagen. Materials and methods FF study samples: • Two FF samples collected from each patient • First follicle punctured bilaterally ‐ without contamination of flushing media, centrifuged (500 x g), supernatant stored at ‐ 20°C. Results 96 patients randomised hCG GnRHa 48 cycles 48 cycles No significant differences between the GnRHa and hCG group: • Age, BMI, base‐line FSH and LH • Infertility diagnosis • Previous IVF/ICSI attempts • Stimulation Aims of study To compare FF levels of amphiregulin in patients undergoing IVF/ICSI following a flexible GnRH antagonist protocol: • Randomisation to final oocyte maturation with either 10.000 IU hCG or 0.5 mg of GnRHa (Buserelin) • Open label prospective randomised three‐centre study • Controls: 15 FF from 12‐15 mm preovulatory follicles prior to ovulation induction 15 FF from small (1‐8 mm) antral follicles (natural cycle) 12 FF from preovulatory follicles ‐ aspirated after endogenous surge (natural cycle) Regulation of oocyte maturation Amphiregulin: EGF‐like peptide produced by the mural granulosa gells but not the cumulus cells Released by the LH‐activity of the mid‐cycle surge Induce resumption of meiosis FSH: Induce yet unidentified substances that promote oocyte maturation (e.g. meiosis activating substance) Amphiregulin LH/hCG Prolactin: Produced by the pituitary, endometrium and granulosa cells PRL inhibits estradiol production and stimulates progesterone production in granulosa cells ± PRL also interferes with FSH action by suppressing LH receptor expression at sites downstream of cAMP synthesis in GC Analysis of PRL knockout mice models has led to a conclusion that PRL is essential for corpus luteum functions Follicular Fluid concentration of E2, P4, Inhibin B, VEGF, Amphiregulin and Prolactin GnRHa vs hCG TOTAL GnRHa hCG t‐test N: OPU 96 48 48 N: FF aspirates 146 73 73 E2, ng/ml 305 ± 17 328 ± 20 282 ± 28 NS P4, ng/ml 13297 ± 466 11758 ± 629 14835 ± 647 0.0004 Inhibin B, ng/ml 27 ± 1.6 25 ± 1.9 29 ± 2.5 NS VEGF, pg/ml 1195 ± 61 1199 ± 83 1192 ± 91 NS Amphiregulin, ng/ml 62 ± 3.5 51 ± 3.5 71 ± 6.0 0.003 Prolactin, ng/ml 17,2 ± 1,9 17,2 ± 2,3 17,3 ± 3,1 NS Data are mean ± SEM Follicular fluid concentration of VEGF and Amphiregulin in controls versus GnRHa and hCG Preovulatory follicular fluid Small antral follicles Before the Natural GnRHa hGG midcycle surge cycle No. FF 15 15 12 73 73 aspirates VEGF, pg/ml 527 ± 517a ND 2248 ± 924b 1199 ± 83c 1192 ± 91c Amphiregulin, 1.5 ± 1.5d < 0.5 68 ± 25e 51 ± 3.5f 71 ± 6.0g ng/ml Groups with a different letter differ significantly (p< 0.003) Data are mean ± SD OOCYTE MATURATION, FERTILIZATION AND EMBRYO DEVELOPMENT Fisher’s Exact TOTAL GnRHa hCG Two-tailed IVF: N: oocyter 685 329 356 N: Fertilized (%) 471 (69%) 237 (72%) 234 (66%) 0.08 IVF: N embryos 405 (86%) 210 (89%) 195 (83%) NS (% of fertilized) ICSI: N: oocyter 145 78 67 N: MII (%) 124 (86%) 72 (92%) 52 (78%) 0.017 N: Fertilized of M II (%) 112 (90%) 61 (85%) 51 (98%) 0.013 ICSI: N embryos 107(96%) 57 (93%) 50 (98%) NS (% of fertilized) IVF + ICSI: Transferrable N (% of oocytes 309 (45%) 167 (51%) 142 (40%) 0.005 retrieved) Transferrable per cycle 3.2 ± 0.3 3.5 ± 0.4 3.0 ± 0.4 NS (mean±SEM) Transferred per cycle 1.65 ± 0.06 1.67 ± 0.07 1.63 ± 0.1 NS (mean±SEM) Triggering ovulation with GnRHa does not compromise embyro implantation rates Acevedo et al., Fertil.
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